P1376: IDENTIFICATION OF SURFACEOME CHANGES IN CD19 CAR-T THERAPY-RESISTANT LYMPHOMA CELLS
Authors: Marta Krawczyk, Klaudyna Fidyt, Katsiaryna Marhelava, Agnieszka Graczyk-Jarzynka, Malgorzata Firczuk, Magdalena Winiarska
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Immunotherapy using T cells expressing chimeric antigen receptor (CAR) targeting CD19 is widely explored and clinically used in the treatment of high-grade hematologic B-cell malignancies. However, despite promising results, many reports indicate that up to 30% of patients develop resistance following that therapy. The knowledge about the resistance development after CD19-targeting CAR-T is still limited as only a few mechanisms underlying this phenomenon have been described. Additionally, the limited access to the relapsed patients’ samples emphasizes the need to develop reliable CD19 CAR-T resistance models that will contribute to a better understanding of the resistant phenotype, as well as can help to identify novel targets and design more efficient immunotherapies. Aims: In this study we aimed to create in vitro models of resistance to CD19 CAR-T therapy and broadly characterize the phenotypic changes in the surfaceome of the resistant cells. Methods: The lymphoma Raji (ATCC) cell line was used to generate two in vitro models of resistance to CD19 CAR-T therapy by long-term repetitive parallel exposure to CD19 CAR-T cells bearing FMC63 scFv, and either CD28 (28z CAR-T) or 4-1BB (BBz CAR-T) costimulatory domain. The CD19 CAR-T cells were produced by lentiviral transduction of primary T cells isolated from buffy coats of healthy donors. The characterization of resistant cells was performed using flow cytometry (FC)-based methods, cytotoxicity assay, multicolor FC panels and Human Cell Surface Marker Screening Panel (BD Lyoplate), and cell surface proteomics. Results: The obtained resistant Raji cells models were verified by CD19 surface status and sensitivity to CD19 CAR-T cells. During the process of resistance generation, we observed that the CD19 loss rate is faster after exposure to 28z CAR-T than to BBz CAR-T cells (Fig. 1A), which corresponds with the clinical observations. Next, we characterized the resistance model obtained with 28z CAR-T cells. Consistent with the clinical observations on the development of resistance to CD19 CAR-T therapy, the resistant Raji cells manifested strong CD19 and CD58 downregulation and a significant decrease in the sensitivity to CD19 CAR-T effectors (Fig. 1B, 1C). Moreover, a multicolor FC analysis of the resistant cells revealed downregulation of many known CD19-interactors and proteins involved in pro-survival pathways in B-cells, like CD21, CD22, CD23, CD40 or CD74. Furthermore, a broad surfaceome analysis of our resistant model using the BD Lyoplate revealed additional changes in the levels of other surface proteins. We observed, among others, the upregulation of CD20 and CD37, and preserved surface level of CD70 and CD38 antigens, all currently tested in clinical trials as targets for CAR-T therapies. Moreover, we used an unbiased approach by performing a biotinylation-based mass spectrometry analysis of cell surface proteins in resistant cells. The analysis confirmed our earlier observations from FC experiments and additionally indicated changes in other molecules, such as CD98 or CXCR4, the role of which is linked with poor prognosis in lymphoma or leukemia patients, but has never been discussed in the context of resistance to CD19 CAR-T therapy.Summary/Conclusion: Our results open doors for a better understanding of the development of resistant mechanisms in tumor cells upon contact with CD19 CAR-T cells. Moreover, they can help exploit specific cellular vulnerabilities in resistant cancer cells and design novel immunotherapeutic schemes as alternatives to CD19 CAR-T therapy for relapse/resistant patients. Keywords: B cell lymphoma, Adoptive immunotherapy, CD19, CAR-T